The menopausal transition is a particularly significant stage in the aging of women, marking the end of fertility and conferring an increased risk of osteoporosis and cardiovascular disease. Genetic determinants of menopausal age can provide insight into the molecular and cellular mechanisms of menopause. One such determinant is the fragile X premutation (i.e. the presence of 55-199 CGG repeats in the 5' UTR of the fragile X mental retardation 1 (FMR1) gene), as 20% of carrier women have premature ovarian failure (POP). While patients with fragile X syndrome have no FMR1 gene expression and a complete absence of fragile X mental retardation protein (FMRP), premutation carriers have an increased level of FMR1 mRNA but a reduced level of FMRP. It is unclear how these molecular events result in premature ovarian failure. One possible mechanism to explain premutation-related POP is altered translational regulation of FOXO3A by FMRP. FOX03A is a known mRNA ligand of FMRP and has been implicated in POF by an analogous phenotype in a knockout mouse model. To determine whether polymorphisms in FOX03A associate with premutation-related POF, we sequenced the promoter and exons of FOXO3A in ten POF subjects (menopausal age <40) and ten subjects of normal menopausal age (menopausal age >46), all of whom carried the fragile X premutation. This sequencing revealed the novel 419T variant allele, in which there is a missense C->T substitution at coding position 419, in three often POF patients but in no subjects with normal age at menopause. One of the POF patients with the FOXO3A 419T variant has an affected sister and daughter who also have the 419T variant, and an unaffected sister lacking this variant, strengthening the evidence of association. We propose to conduct a case-control study to more completely characterize the association between fragile X premutation-related POF and the FOXO3A 419T variant. To define whether the association between POF and the variant is causal, we propose to create a transgenic mouse model of the FOXO3A 419T variant. Characterization of the reproductive physiology of this mouse will provide insight into how the FOXO3A 419T variant impacts fertility in humans. The 419T variant causes an alanine to valine substitution at amino acid 140, a residue conserved in mammals, and therefore is likely to alter FOXO3A function. We propose to characterize the molecular effect of the FOXO3A 419T variant. Through characterization of the FOX03A 419T variant, we can more fully understand a potentially important genetic risk factor for premature ovarian failure. The results of this research will allow women at risk for POF to be identified early, and will yield a more complete understanding of menopause and female fertility. [unreadable] [unreadable] [unreadable]